The present invention is directed to a process to analyze single nucleotide polymorphisms (SNPs) in highly polymorphic nucleic acids, particularly in those targets that contain one or more additional polymorphisms close to the SNP of interest.
Typically genotyping of a single nucleotide polymorphism is performed using a probe or a primer containing a single mismatch. This simplifies the analysis of alleles tremendously. However, the presence of more than one polymorphism in a probe region complicates genotyping and makes detection very difficult if not impossible for many SNP detection methods. Most SNP detection methods present difficulties in genotyping a polymorphism of interest if the probe sequence spans another polymorphism.
Single nucleotide polymorphisms (SNPs) are the most abundant of variations in the human genome, accounting for >90% of sequence polymorphisms. They occur on average once every 1000 nucleotides so that, as a rule of thumb, there is a 0.1% chance of any base position being heterozygous in a particular individual. Nucleotide diversity is not constant over the entire genome, with areas of extremely low diversity, for example, the X chromosome, and areas of extremely high diversity, for example, up to 10% in the human leukocyte antigen (HLA) loci (Twyman and Primrose, Pharmacogenomics 4: 1-13 (2002)). Highly polymorphic SNP target sequences are also known in other species, e.g., plants (Ching A, Caldwell K S, Jung M, Dolan M, Smith O S, Tingey S, Morgante M, Rafalski A J. BMC Genet. 3: 19 (2002)) and viruses (Miller V, Larder B A. Antivir Ther. 6 Suppl 3:25-44 (2001)). Many SNP detection methods have been described to detect a single polymorphism in a nucleic acid target in absence of another polymorphism in close proximity (Twyman and Primrose, supra; Gerome Breen, Psychiatric Genetics, 12: 83-88 (2002)). Genotyping by melting curve analysis has been developed (see, U.S. Pat. Nos. 6,569,627; 6,506,568; 6,448,015 and U.S. application Ser. No. 10/165,410, each of which is hereby incorporated herein by reference).
Others have not designed probes or used universal bases to identify a polymorphism of interest when a second polymorphism resides within the length of the probe, such that the probe incorporates a universal base in a location to pair with the second polymorphism. For instance, in a summary of applications for universal base analogues, David Loakes, Nucl. Acids Res., 29: 2437-2447 (2001), discusses SNP analysis, but not when more than one polymorphism is covered by the probe. Bergstrom et al, J. Amer. Chem. Soc., 117: 1201-1209 (1995) disclose the synthesis of 1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole. The melting temperatures (Tms) of 15-mer oligonucleotides containing a single universal base hybridized to a complement containing either A, C, G or T at that position were within 3° C. However, the Tms were significantly lower than that of fully matched oligonucleotides. Guo et al, (Nat. Biotech., 15: 331-335 (1997) and U.S. Pat. No. 5,780,233)), disclose the use of 3-nitropyrrole to increase mismatch discrimination by the introduction of this base as an artificial mismatch. U.S. Patent Publication 2003/0165888 discloses probe and primers that contain at least two juxtaposed universal bases. Bowden et al (Thromb. Heamost., 80: 32-36 (1998)) disclose the simultaneous detection of multiple SNPs using a probe that contains a microdeletion or microinsertion of natural bases in the vicinity of established mutation sites. WO 03/040395 discloses universal nucleotides referred to as extendable nucleotides that can be incorporated into a polynucleotide strand by a polymerase during a primer extension reaction. EP 1314734 discloses a limited number of LNA substituted phosphoramidite reagents. WO 02/062816 discloses a large number of universal base triphosphate derivatives for pharmaceutical applications.
Several publications disclose universal bases without discussing their application in SNP analysis. For example, Lin, P K T and Brown, D M, Nucl. Acids Res., 20: 5149-5152 (1992) disclose two new degenerated bases that can be used in probes and primers, 6H,8H-3,4-dihydropyrmido[4,5-c][1,2]oxazin-7-one (P) and 2-amino-6-methoxyaminopurine (K), but do not disclose their use in mismatch discrimination or genotyping. Due to their ability of both base P and base K to exist in their amino and imino tautomers, they base pair with both A and G, and C and T, respectively. McMinn et al, J. Amer. Chem. Soc., 121: 11585-11586  (1999) and Ogawa et al, J. Amer. Chem. Soc, 122, 3274-3287 (2000) disclose the synthesis of unnatural nucleobases based on an isocarbostyryl core structure. Matsuda et al, J. Amer. Chem. Soc., 125: 6134-6139 (2003), disclose the synthesis of four unnatural bases, each of which hybridize with all four natural bases relatively similarly. Kim et al, Biorg. Med. Chem. Let., 12: 1977-1980 (2002) disclose the base pair properties of 8-oxo-7,8-dihyroadenosine. Henry et al, J. Amer. Chem. Soc., 125: 9638-9646 (2003) disclose six new unnatural bases, but did not investigate SNP analysis. Seela and Debelak (Nucleosides, Nucleotides & Nucl. Acids, 20: 577-585 (2001); Seela and Debelak (Nucl. Acids Res., 28: 3224-3232 (2000) and WO 01/72764 disclose three universal bases which showed no significant structural discrimination with natural analogs A, C, G and T. EP 1314734 discloses a limited number of LNA substituted phosphoramidite reagents. WO 02/062816 discloses a large number of universal base triphosphate derivatives for pharmaceutical applications.
Nucleic acid target amplified-based analyses are disclosed in U.S. Pat. Nos. 4,800,159; 6,197,563; 5,210,015; 5,312,728; and 6,221,603, each of which is hereby incorporated by reference.
Natural modified base analogs and their properties have been disclosed in WO 01/65958, U.S. Pat. Nos. 6,127,121; 6,660,845; and RE 38,416 and Seela et al, Helv. Chim., Acta, 69:1602-1613 (1986) each of which is incorporated herein by reference.
Accordingly, there is a need for primers/probes that can distinguish a first SNP of interest that is located within the length of a probe of a second SNP, and methods for designing and using such primer/probes. The present invention fulfills this and other needs.